Turbidity is basically how cloudy a liquid is. For example, vodka has a low turbidity, but milk has a high turbidity.

For our goal of genotyping salmon, we decided to use LAMP (loop-mediated isothermal amplification) because of its ability to give a cheap and easy binary result (yes, it’s coho, or no, it isn’t coho).

[This method uses a DNA polymerase with strand displacement activity and yields large quantities of DNA in less than an hour under constant temperature (thus obviating the need for expensive thermocyclers). Moreover, a correspondingly large amount of white magnesium pyrophosphate precipitate is synthesized as a by-product, enabling direct visual confirmation of amplification success through monitoring of mixture turbidity. These characteristics provide results in a fast, efficient, and cost-effective method that theoretically should amplify target sequences with higher specificity than PCR.] – https://www.nature.com/articles/srep40125

From the papers we reviewed, we learned that a positive LAMP amplification would result in the production and precipitation of magnesium pyrophosphate (Mg2P2O7). We’ve been designing based on the idea that we would be able to see the change in turbidity during a positive control. Unfortunately, we learned later that the change in turbidity would not be visual to the naked eye (change in turbidity would be around 0.7 NTU….basically invisible).

Citizen salmon is still here and progressing with LAMP. We had talked about LAMP a bunch in the past, but are finally making progress despite some rough starts.

Recap:

We had chosen to move forward with LAMP because its isothermal (so light on the equipment needs) and suppose to result in visible-turbidity if positive (no equipment necessary for analysis).

Unfortunately with the salmon primers we had designed we weren’t getting any visible turbidity after 1-2 hours of incubation. We would leave our samples incubating overnight, then add SYBRsafe to the tubes to see if any DNA amplified. This is what we got. All positives and negatives growing like crazy!

We think that our primers are self-amplifying, which really brings our primer-designing ability into question. So what next?

Well, lets test our primer design skills with an easier target. At SoundBio, we have a circular piece of DNA called pPSU1 that can be cut to various sizes to form a DNA ladder. We thought lets cut the circular DNA once to make it linear and practice primer design the pPSU1 DNA.

For this week’s Salmon Night we picked up on the nucleic acid precipitation protocol we began last week and explored another technique for genomic DNA extraction.

Background

Genomic DNA extraction from tissue has three main steps. The first step is typically disrupting the tissue and breaking open the membrane of cells. The second step is the removal of containments and unwanted cellular material (typically proteins and lipids). And finally, recovering and purifying the DNA from solution.

In the Chelex extraction procedure, the chelex resin themselves, along with boiling, help disrupt the tissue. The chelex resin also acts via ion exchange to trap Mg++ ions in the sample. Mg++ is a necessary cofactor for DNAases, or enzymes that degrade DNA, so by removing them from the solution, potential DNAases are inactivated. Centrifugation is then used to clarify the solution. Here is the protocol we have been using.

Other methods for extraction include a using a combination of a detergent, a proteinase, a chelating agent, nonpolar alcohols and salts. The detergent helps remove lipids (think oils), the proteinase helps degrade unwanted protein contaminants, the chelating agent traps the DNAase cofactor Mg++ and the nonpolar alcohols and salts help precipitate, and thereby purifiy, the DNA after centrifugation.

Protocol

Solution after step 1

To make the genomic DNA extraction solution we followed the recipe made by a mouse genotyping lab (Tsai Lab in the links below)

A little late but here is an update on the work that Bri and I worked on last Tuesday. Picking up from the chelex extraction that Carson and Allison performed and the chelex Proteinase-K extraction that Anna performed, our goal for the night was to set up and hopefully run a nucleic acid precipitation of their extractions. While we can use the DNA extracted from the chelex protocol in downstream applications like PCR, the resin in the chelex prevents accurate absorbance-based DNA quantification. Our hypothesis is that the nucleic acid precipitation would help purify the DNA and allow us to use the donated spectrophotometer we have in lab currently as a quality control check.

Background

This protocol works by relying on the fact that DNA is slightly polar. The slightly polar charge of DNA causes DNA to be less soluble in non-polar alcohol than they are in polar water. Additionally, the phosphate-sugar backbone of DNA, which is responsible for DNA’s slight negative charge, can be neutralized with the addition of salt making the DNA less hydrophilic and thus more able to precipitate.

Gel Electrophoresis of Sunday’s PCR Product (Tues 02.28.17)

Today we ran two gels to see what we got from Sunday’s PCR. Because we had limited time Emily Chen and I can two gels. Our sample names are based on the PCR tubes.

Made 1.5% Agarose gels (3g agarose in 200ml 1x TAE)

Add SYBRsafe to gels.

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I (Regina 🙂 ran a small gel with A3-A9. Emily Chen ran the larger gel with C3-C11.

We used a NEB Ready-load 100bp ladder and a Qiagen sample loading buffer. Thank-you to Riti for making the gel sheets to help with recording. (Being an idiot I forgot to log my run time. It was ~30min.)

Results

I was able to get faint smearing bands for A3-A9. The different between these samples was the annealing temperatures. The wells for this gel were very large for a small gel. I think this might be why the bands are not very crisp; however the ladder turned out great.

Emily’s gel (C3-C11) contained no bands except for the ladder (no picture). Thus primer set 3 might be the problem.

Michal Galdzicki, Chinook Science Officer (CSO) of SoundBio, just presented at NerdNite in Seattle! In front of an audience of kindred nerds, our chinook swam upstage to talk about our hopes and dreams for the future of DIY salmon genotyping. We hope that 2017 will be a new run for us with an active salmon cohort, a home river in U-District, and new donations and ideas to help us build momentum. Thank to everyone that was able to attend and to NerdNite for being such an awesome venue to talk about our project!

Expert writing and stellar personality skills paid off as our abstract was accepted and we presented at BioHACK THE PLANET’s conference that was held in Oakland, CA last month. You know what else paid off? Above average artistry. We were the best dressed, hands down. Watch our Chinook, Mike, give an excellent talk dressed as, well, a Chinook. What else would it be? He’s king!